Oxidized Phospholipids as Endogenous Pattern Recognition Ligands in Innate Immunity

One of the major functions of the innate immune system is the surveillance of host tissues, identifying apoptotic and senescent cells for engulfment and orderly removal by macrophages (1, 2). Macrophage recognition of modified lipoproteins occurs via similar pathways of the innate immune system, involving shared scavenger receptors and molecular pattern recognition ligands (3–6). These critical homeostatic functions of the innate immune system are essential to a diverse array of physiological processes, ranging from embryonic development and tissue remodeling to resolution of inflammation (7, 8).

Phagocytic cells express a broad range of receptors that participate in recognition and engulfment of apoptotic and senescent cells, including CD36, a prototypic member of the class B scavenger receptor family (9–14). CD36 recognizes pathogen-associated molecular patterns, including erythrocytes infected with Plasmodium falciparum (15, 16). It also recognizes oxidatively modified lipoproteins, leading to cholesterol accumulation and atherosclerotic plaque development in vivo (17, 18). Recent data support the notion that phospholipid oxidation within cell membranes and lipoproteins exposes similar molecular patterns recognized by CD36 and other receptors of innate immunity. Animal model studies with mice genetically engineered to lack functional CD36 confirm that this prototypic scavenger receptor participates in recognition and engulfment of apoptotic cells (19), senescent cells or cell fragments (20), and oxLDL2 in vivo (18, 21).

This review will discuss recent discoveries regarding the structural nature of oxidized phospholipids that serve as high affinity ligands for CD36 in vivo. Also discussed will be the Lipid Whisker Model (22). A revision to the Fluid Mosaic Model (23), the Lipid Whisker Model focuses on phospholipid conformation within oxidized cell membranes and lipoproteins and is derived from recent studies demonstrating the generality of a conformational switch in the structure of many phospholipids within cell membranes that occurs following oxidation. The conformational switch, involving the protrusion of oxidized fatty acids from the hydrophobic membrane interior into the more polar aqueous compartment (22, 24), facilitates physical contact between pattern recognition receptor and molecular pattern ligand. This anatomic positioning of oxidized fatty acids of membrane phospholipids may also underlie the preferential selectivity of some phospholipases for oxidized fatty acids during membrane remodeling.

oxPC within the Lipid Component of oxLDL and Oxidized Membranes Plays a Major Role in Macrophage Recognition and Phagocytosis via CD36

Recognition of oxidatively modified lipids on the surface of cell membranes and lipoprotein particles plays an important role in numerous innate immune functions and can trigger diverse cellular processes (25–32). Over the past several years, much effort has focused on our understanding of the chemical and structural nature of oxPL ligands recognized by macrophage pattern recognition receptors. There is general consensus that a significant portion of ligand binding activity on oxidized lipoproteins and senescent or apoptotic cells resides within extractable lipids (33–37). For example, studies from the collaborative research groups of Steinberg and Witztum (35) first reported that receptors for oxLDL on elicited mouse peritoneal macrophages recognize both the oxidized lipid component and the modified protein moieties of oxLDL. In an extension of those studies, these investigators later identified the mouse scavenger receptor CD36 responsible for mediating binding to copper oxLDL via oxPC (34). In competition studies ∼50% of the CD36 binding activity was shown to reside within the lipid (organic solvent)-extractable fraction (34). Concurrently, Podrez et al. (33) reported results from studies employing LDL exposed to the myeloperoxidase-H₂O₂-nitrite system of monocytes, a more physiologically relevant oxidized form of LDL. Here, the vast majority of macrophage recognition of the oxidatively modified lipoprotein was shown to reside within the lipid-extractable portion of the particle, with >90% of the binding attributable to oxPC interaction with macrophage scavenger receptor CD36 (33).

Peptide-bound oxPC Also Plays a Role in Macrophage Recognition and Phagocytosis of Oxidatively Modified LDL and Cell Membranes

In studies with copper oxLDL, at least half of the macrophage binding activity was observed to remain in the protein moieties of lipoprotein following solvent extraction, suggesting that protein-oxPL adducts may also serve as ligands for macrophage recognition and phagocytosis via CD36 (34). This has also been observed in model studies employing protein-lipid and peptide-lipid adducts. Based upon studies with model protein- or peptide-oxPL adducts generated in vitro, a role for macrophage scavenger recognition of the choline headgroup of glycerophospholipids when tethered to proteins has also been reported (34, 38, 39). Alternative members of the innate immune system such as the acute-phase reactant C-reactive protein (38) and antibodies to protein-oxPC adducts have been shown to bind to both oxLDL and apoptotic cells through recognition of oxPC as a common ligand (5, 38).

Structural Characterization of oxPC That Serves as a High Affinity Ligand for Macrophage CD36

Substantial progress has been made over the last several years in delineating structures of oxidized diradyl glycerophospholipids that facilitate macrophage recognition of oxLDL and oxidized, senescent, and apoptotic cell membranes. Podrez et al. (40, 41) reported the first systematic study aimed at identifying the structures of specific oxidized lipids that serve as ligands for the scavenger receptor CD36. Structure/function analyses with multiple synthetic species defined critical structural elements of endogenous ligands for CD36 that promote high affinity binding to the scavenger receptor: a phospholipid with a truncated sn-2 acyl group that incorporates a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl (oxPC_CD36) (40, 41). The structures of the oxidized lipid ligands of CD36 are illustrated in Fig. 1A.

FIGURE 1.

Structures of identified oxPL that serve as ligands for the scavenger receptor CD36 (oxPL_CD36) within membranes. A, the length of the oxidized truncated sn-2 acyl chain varies depending upon the parent (unoxidized) fatty acid precursor, with n = 2, 3, and 7 for docosahexaenoic, arachidonic, and linoleic acids, respectively. The structural motif within the dashed box confers high affinity CD36 recognition when tethered to the sn-2 acyl group of a phospholipid. HODA-PL, HOOA-PL, and HOHA-PL, the 9-hydroxy-12-oxododec-10-enoic acid, 5-hydroxy-8-oxoocta-6-enoic acid, and 4-hydroxy-7-oxohept-5-enoic acid esters of 1-palmitoyl-2-lyso-sn-3-glycerophospholipid, respectively; KODA-PL, KOOA-PL, and KOHA-PL, the 9,12-dioxododec-10-enoic acid, 5,8-dioxooct-6-enoic acid, and 4,7-dioxohept-6-enoic acid esters of 1-palmitoyl-2-lyso-sn-3-glycerophospholipid, respectively; HDdiA-PL, HOdiA-PL, and HHdiA-PL, the 9-hydroxy-11-carboxyundec-6-enoic acid, 7-carboxy-5-hydroxyhept-6-enoic acid, and 4-hydroxy-7-carboxyhex-5-enoic acid esters of 1-palmitoyl-2-lyso-sn-3-glycerophospholipid; respectively; KDdiA-PL, KOdiA-PL, and KHdiA-PL, the 11-carboxyl-9-oxoundec-6-enoic acid, 7-carboxy-5-oxohept-6-enoic acid, and 6-carboxy-4-oxohex-5-enoic acid esters of 1-palmitoyl-2-lyso-sn-3-glycerophospholipid, respectively; PA, phosphatidic acid; PDHA, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-PC. B, structures of select additional oxidation products derived from oxPL_CD36 containing sn-1 palmitic acid and sn-2 linoleic acid (left), arachidonic acid (middle), or docosahexaenoic acid (right) are shown. ON-PL, OV-PL, OB-PC, A-PL, G-PL, and S-PL, the 9-oxononanoic acid, 5-oxovaleric acid, 4-oxobutyric acid, azeleic acid, glutaric acid, and succinic acid esters of 1-palmitoyl-2-lyso-sn-3-glycerophospholipid, respectively; Z, phospholipid headgroup. n = 7, 3, and 2 for linoleic, arachidonic, and docosahexaenoic acids, respectively.

Although initially defined using PC molecular species, as outlined below, subsequent studies have shown that all classes of phospholipids examined thus far (PC, phosphatidylethanolamine, PS, and phosphatidic acid) harboring the high affinity motif for CD36 promote CD36-mediated recognition and phagocytosis (19, 20, 22, 40, 41). The structures of oxPC_CD36 identified were established using a combination of cell binding studies and multiple distinct chromatographic and MS methods in conjunction with (i) results of numerous derivatization strategies to ascertain functional groups on isolated products, (ii) inference of structures of products that appeared plausible based upon MS results and known mechanisms of lipid oxidation and fragmentation, and (iii) de novo synthesis of each oxidized lipid. The identities of the oxidation products and synthetic standards were confirmed by demonstration that synthetic species recapitulate all biological, chemical, MS, and chromatographic characteristics of the lipids isolated from oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-PC, oxidized 1-palmitoyl-2-lineoyl-sn-glycero-3-PC, and oxidized 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-PC (19, 20, 22, 40, 41). Liquid chromatographic/electrospray ionization/tandem MS analyses have demonstrated the generation of these species in lipoproteins oxidized by multiple distinct pathways in vitro (40), in atherosclerotic lesions in vivo (41), in retina following light exposure in dark-adapted rodents (20), and in cerebral tissues following ischemia-reperfusion injury (42). Fig. 1B illustrates structures of additional oxidized glycerophospholipids detected in the above in vivo models that have been shown to be generated by further oxidative fragmentation of oxPL_CD36. Although many have been reported to possess pro-inflammatory biological activities (32) or when adducted to proteins may mediate scavenger receptor recognition (34), they do not bind with high affinity to CD36 when present in membranes as free oxPL (40, 41).

Formation and Decay of oxPL_CD36

An overall scheme illustrating potential pathways responsible for formation and decay of oxPL_CD36 is illustrated in Fig. 2. In vitro studies confirm the capacity of multiple distinct oxidant pathways to generate oxPL_CD36 (40). The pathways shown represent known initiators of lipid peroxidation (e.g. myeloperoxidase, lipoxygenases, reactive nitrogen species) or known sources of reactive oxidant species (NAD(P)H oxidase, cytochrome c). Indeed, recent studies by Kagen and co-workers (43, 44) have reported that cytochrome c-mediated oxidation and externalization of PS during apoptosis facilitate phagocyte uptake of apoptotic cells. Thus, oxPL_CD36 are formed following initiation of lipid peroxidation, regardless of whether triggered by inflammation, senescence, or an apoptogenic trigger. The appearance of oxPL_CD36 within membranes can then promote phagocyte recognition and engulfment, even when present at only a few molecules per particle (Fig. 2) (41).

FIGURE 2.

Scheme of potential pathways impacting upon formation and decay of oxPL that serve as high affinity ligands for the scavenger receptor CD36. CytoC, cytochrome c; RNS, reactive nitrogen species; MPO, myeloperoxidase; NOX, NAD(P)H oxidase; LPO, lipoxygenase; PAF-AH, platelet-activating factor acetylhydrolase; OB-PL, ON-PL, and OV-PL, the 4-oxobutyric acid, 9-oxononanoic acid, and 5-oxovaleric acid esters of 2-lyso-PC, respectively; S-PL, A-PL, and G-PL, succinic acid, azeleic acid, and glutaric acid esters of 2-lyso-PC, respectively. oxPL_CD36 binds to the scavenger receptor CD36. oxPC-furan is oxPL with an sn-2 acyl group that incorporates a terminal 2-furyl carbonyl. This figure was reprinted from Ref. 42.

Although one fate of oxPL_CD36 may be phagocyte uptake, alternative potential destinies for the oxidized lipids clearly also exist (Fig. 2). For example, further oxidation leads to formation of more terminally oxidized and truncated species, which can facilitate myriad potential downstream signaling pathways. Alternatively, the oxidized lipids may serve as targets for phospholipase activity, such as for platelet-activating factor acetylhydrolase (Fig. 2) (45, 46). This PLA₂ selectively cleaves sn-2 fatty acids from oxPC species, promoting membrane remodeling and clearance of oxPL species. A subset of oxPL_CD36 are reactive electrophilic species, capable of covalently modifying nucleophilic targets such as various residues of proteins and amino lipids. Finally, a subset of oxPL_CD36, molecular species possessing reactive esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPL species possessing an sn-2 acyl group that incorporates a terminal furyl moiety (oxPL-furan) (Fig. 2) (42). In contrast to their precursors, oxPC-furans do not bind to CD36 and fail to trigger phagocytosis (42). Using high pressure liquid chromatography with on-line tandem MS in combination with unambiguous organic synthesis, oxPC-furans ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids have been detected following exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress such as within brain tissues following cerebral ischemia has also been reported (42).

oxPS (Not Non-oxPS) Serves as a Recognition Ligand for Macrophage-dependent Phagocytosis of Apoptotic Cells via CD36

The externalization of PS on the exofacial leaflet of the plasma membrane in response to multiple apoptogenic triggers is widely held as an enabling event for promoting phagocyte recognition and engulfment of apoptotic cells by macrophages. The scavenger receptor CD36 figures prominently among the scavenger receptors implicated as participants in apoptotic cell engulfment via PS recognition as a ligand (13, 14). Moreover, transfection of cells with human CD36 has been shown to confer professional phagocytic function to otherwise non-phagocytic cell types (13). Recent studies using CD36 knock-out mice confirm a role for CD36 in apoptotic cell clearance in vivo because mice with CD36 deficiency demonstrate impaired ability to efficiently clear apoptotic cells in an inflammatory wound injury model (19).

Despite the “dogma” supporting PS as the major lipid ligand triggering apoptotic cell recognition, a growing body of evidence supports an alternative notion. Over a decade ago, Steinberg and co-workers reported that recognition of oxidatively damaged and apoptotic cells by macrophages occurs by receptors that similarly recognize oxLDL (6) and that oxidation epitopes recognized by these macrophage receptors reside within both lipid-extractable and protein components of oxLDL (35–37). Witztum and co-workers (5) further confirmed that oxidation-specific epitopes on apoptotic cells cross-react with multiple antibodies specific for oxPL and, through competition studies, showed that oxidized lipids on the apoptotic cell surface serve as ligands for recognition and phagocytosis by macrophages. Recently, Kagen and co-workers (43, 44, 47) provided evidence to support the concept that selective oxidation of PS occurs during apoptosis, presumably via peroxidase activity of mitochondrial cytochrome c, promoting PS externalization possibly by altering aminophospholipid translocase activity. Finally, in a recent series of studies, Greenberg et al. (19) demonstrated that CD36-specific binding and uptake of PS-containing vesicles are mediated by oxPS (not non-oxPS) species. A critical role for oxPS species in apoptotic cell recognition by macrophage CD36 was supported by multiple independent lines of evidence. Liposomes containing oxPS (but not non-oxPS) were shown to preferentially bind to CD36-transfected cells, whereas complementary studies employing wild-type and CD36 knock-out peritoneal macrophages confirmed a requirement for oxidation of PS for cell binding and phagocytosis via endogenously expressed CD36 in macrophages (19). Furthermore, incorporation of oxPS (but not PS) into viable non-apoptotic cell membranes was shown to confer CD36 binding activity and capacity to be phagocytosed by CD36-bearing cells. Finally, studies employing multiple distinct cells and apoptotic triggers confirmed a critical role for oxPS versus PS as a recognition ligand for CD36-mediated phagocytosis, with multiple MS-based approaches showing that oxPS species possessing the structurally conserved CD36 recognition motif are formed within apoptotic membranes and thus may play a role in CD36-dependent recognition of apoptotic cells (19).

The Lipid Whisker Model

Recent studies into the conformation of oxPL species recognized by CD36 within model membranes (22, 24) have led to the development of the Lipid Whisker Model (22), a refinement to the classic Fluid Mosaic Model first proposed by Singer and Nicolson (23). A key aspect of the Fluid Mosaic Model is that amphipathic phospholipids are oriented into a lamellar mesophase organization, with hydrophobic fatty acyl chains buried within the membrane interior and hydrophilic polar headgroups oriented toward the aqueous milieu. This lipid organization permits rapid lateral diffusion of lipid and transmembrane protein alike within the planar membrane surface. It also underlies the impermeant nature of cell membranes to hydrophilic species. X-ray studies confirm this structural organization of lipids within cell membranes. However, recent data suggest that in peroxidized cell membranes, many of the oxPL species adopt a different conformation.

In the Lipid Whisker Model, the orientation of many oxPL within cell membranes differs from the classic architecture of neighboring non-oxPL. Biophysical studies have revealed the conformation of structurally defined synthetic oxPC within perdeuterated membranes. Addition of a polar oxygen atom to numerous peroxidized fatty acids reorients the acyl chain whereby it no longer remains buried within the membrane interior, but rather protrudes into the aqueous compartment (Fig. 3) (22, 24). This remarkable conformational change enables direct physical access of the oxidized fatty acid moiety to cell-surface scavenger receptors on scanning macrophages. The conformation of individual lipids was inferred by determining multiple critical internuclear distances using nuclear Overhauser effect spectroscopy of individual structurally defined phospholipid molecular species within perdeuterated lipids in model membrane bilayers (22, 24). The conformation of the lipids thus revealed suggests the following global phenomenon: as cell membranes undergo lipid peroxidation, such as during inflammation, senescence, or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of lipid bilayers to the aqueous exterior. This conformational shift may enable physical contact between pattern recognition receptor and molecular pattern ligand. From the vantage of the cell surface, membranes will apparently “grow whiskers” as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface (Fig. 4).

FIGURE 3.

Comparison of the structure of lamellar phase PC (e.g. dimyristoyl-PC; b) and the predicated conformation of 1-palmitoyl-2-(5-keto-6-octenedioyl)-PC (a) within membranes. a, the structure of 1-palmitoyl-2-(5-keto-6-octenedioyl)-PC (KOdiA-PC), a prototypic oxPC_CD36, is shown. b, the conformation of dimyristoyl-PC (DMPC) was redrawn according to reported x-ray diffraction studies. This figure was reprinted with permission from Li et al. (24).

FIGURE 4.

Schematic representation of the Lipid Whisker Model. Cell membranes of senescent or apoptotic cells possess oxPL with a variety of oxidized fatty acyl chains of differing structures protruding into the aqueous compartment. This conformation renders them accessible to interact with scavenger receptors and other pattern recognition receptors on the surface of probing macrophages of the innate immune system. It may also render them more accessible for some phospholipases, promoting membrane remodeling as oxidized fatty acids are cleaved and lysophospholipids reacylated. This figure was reprinted from Ref. 22.

The conformational shift in a fatty acyl chain upon oxidation within a membrane bilayer may serve as the triggering event for numerous downstream biological activities. Most important is likely the rendering of structurally specific and important functional groups of biological signaling molecules into an anatomic location on the cell surface where receptor engagement and subsequent signaling may occur. For example, CD36 recognition of specific oxPL may not only trigger macrophage phagocytosis (13, 14, 41), foam cell formation, or other intracellular signaling cascades within macrophages (21, 33), but also has been linked to alteration in platelet reactivity and in vivo rates of thrombosis (29). The platelet-activating factor receptor similarly recognizes numerous oxPC species with truncated oxidized fatty acid groups at the sn-2 position (48, 49), species that will possess oxidatively truncated fatty acyl groups protruding into the aqueous compartment (22). The multiple signaling events triggered by engagement of the platelet-activating factor receptor with these oxPC species are likely facilitated by the unusual conformational orientation of the oxidized fatty acid chains on these oxPC species. It also is of interest that some PLA₂ enzymes have been reported to demonstrate selective substrate preference for oxPL (45, 46). It is tempting to speculate that the substrate specificity of these PLA₂ enzymes is related to the conformational orientation of the oxidized sn-2 fatty acid species within a membrane bilayer.

Tremendous advances have been made in defining the diverse molecular species of lipids that exist within a membrane and the complexities of their organizational structure. Membrane lipid peroxidation can generate a seemingly bewildering array of possible species, the structures and conformations of which play important roles in normal and disease pathobiology. Nature has apparently exploited this rich and diverse canvas for innate immune recognition engagement and signaling pathways. Indeed, structurally important chemical groups and conformations appear to serve as shared molecular patterns for detection of aged and oxidatively modified cells, lipoproteins, apoptotic cells, and cellular debris. Similar patterns appear on the surface of pathogens, merging innate immune functions of host defense with homeostasis surveillance functions. oxPL represent surface-accessible structurally defined entities that carry chemical and pattern recognition signals, a virtual “Braille” system for molecular pattern recognition as illustrated in the Lipid Whisker Model. Further studies are needed to learn how to “read” better both chemical and conformational information inscribed within individual “whiskers” on the surface of peroxidized membranes and lipoproteins and how their cognate receptors engage them.